Early Life Represents a Vulnerable Time Window for IL-33-Induced Peripheral Lung Pathology.

IL-33, an IL-1 family cytokine, is constitutively expressed in mucosal tissues and other organs in healthy humans and animals, and expression levels increase in inflammatory conditions. Although IL-33-mediated promotion of type 2 immune responses has been well established, a gap in our knowledge regarding the functional diversity of this pleiotropic cytokine remains. To address this gap, we developed a new IL-33 transgenic mouse model in which overexpression of full-length IL-33 is induced in lung epithelial cells under conditional control. In adult mice, an ∼3-fold increase in the steady-state IL-33 levels produced no pathologic effects in the lungs. When exposed to airborne allergens, adult transgenic mice released more IL-33 extracellularly and exhibited robust type 2 immune responses. In neonatal transgenic mice, up to postnatal day 14, a similar increase in steady-state IL-33 levels resulted in increased mortality, enlarged alveolar spaces resembling bronchopulmonary dysplasia, and altered expression of genes associated with tissue morphogenesis. Processed 25-kDa IL-33 protein was detected in bronchoalveolar lavage fluids without any exogenous stimuli, and pathologic changes were abolished in mice deficient in the IL-33 receptor ST2. These findings suggest that adult lungs are relatively resistant to IL-33 overexpression unless they encounter environmental insults, whereas developing lungs are highly susceptible, with IL-33 overexpression resulting in detrimental and pathologic outcomes.

B cells play key roles in th2-type airway immune responses in mice exposed to natural airborne allergens.

Humans are frequently exposed to various airborne allergens. In addition to producing antibodies, B cells participate in immune responses via various mechanisms. The roles of B cells in allergic airway inflammation and asthma have been controversial. We examined the functional importance of B cells in a mouse model of asthma, in which mice were exposed repeatedly to common airborne allergens. Naïve wild-type BALB/c mice or B cell-deficient JH-/- mice were exposed intranasally to a cocktail of allergen extracts, including Alternaria, Aspergillus, and house dust mite, every other day for two weeks. Ovalbumin was included in the cocktail to monitor the T cell immune response. Airway inflammation, lung pathology, and airway reactivity were analyzed. The airway exposure of naïve wild type mice to airborne allergens induced robust eosinophilic airway inflammation, increased the levels of Th2 cytokines and chemokines in the lung, and increased the reactivity to inhaled methacholine. These pathological changes and immune responses were attenuated in B cell-deficient JH-/- mice. The allergen-induced expansion of CD4+ T cells was impaired in the lungs and draining lymph nodes of JH-/- mice. Furthermore, lymphocytes from JH-/- mice failed to produce Th2 cytokines in response to ovalbumin re-stimulation in vitro. Our results suggest that B cells are required for the optimal development of Th2-type immune responses and airway inflammation when exposed to common airborne allergens. The therapeutic targeting of B cells may be beneficial to treat asthma in certain patients.

An immunologic test for chronic rhinosinusitis based on free intranasal eosinophilic major basic protein.

BACKGROUND: A histologic hallmark of chronic rhinosinusitis (CRS) is an eosinophilic inflammation, present with and without nasal polyposis and independent of atopy. Eosinophils migrate through nasal tissue including the epithelium into the nasal airway mucus, where they form clusters and degranulate, releasing granule proteins including the toxic major basic protein (MBP). Specific biomarkers for CRS, which could be used as a diagnostic test for CRS with a high sensitivity and specificity, are presently lacking. Recently, an enzyme-linked immunosorbent assay (ELISA)-based test for MBP in nasal airway mucus received regulatory approval. METHODS: A new assay was specifically developed to detect released MBP in airway mucus. MBP levels in nasal mucus of 85 randomly selected CRS patients diagnosed by endoscopy, computed tomography (CT) scans and symptoms were compared to 13 healthy controls and 5 disease controls (allergic rhinitis). RESULTS: Overall, 92% (78/85) of CRS patients' mucus were positive for MBP (mean 7722 ng/mL) vs none of 13 healthy controls and none of 5 allergic rhinitis patients (<7.8 ng/mL; p < 0.000000000002). In this study, the MBP ELISA had a 92% sensitivity and 100% specificity for CRS. CONCLUSION: Free MBP in nasal mucus can be used as a biomarker to diagnose CRS. The MBP ELISA represents the first immunologically-based test to potentially distinguish CRS from the eosinophilic inflammation in allergic rhinitis.

Accuracy, safety, and tolerability of tissue collection by Cytosponge vs endoscopy for evaluation of eosinophilic esophagitis.

BACKGROUND & AIMS: Management of eosinophilic esophagitis (EoE) requires repeated endoscopic collection of mucosal samples to assess disease activity and response to therapy. An easier and less expensive means of monitoring of EoE is required. We compared the accuracy, safety, and tolerability of sample collection via Cytosponge (an ingestible gelatin capsule comprising compressed mesh attached to a string) with those of endoscopy for assessment of EoE. METHODS: Esophageal tissues were collected from 20 patients with EoE (all with dysphagia, 15 with stricture, 13 with active EoE) via Cytosponge and then by endoscopy. Number of eosinophils/high-power field and levels of eosinophil-derived neurotoxin were determined; hematoxylin-eosin staining was performed. We compared the adequacy, diagnostic accuracy, safety, and patient preference for sample collection via Cytosponge vs endoscopy procedures. RESULTS: All 20 samples collected by Cytosponge were adequate for analysis. By using a cutoff value of 15 eosinophils/high power field, analysis of samples collected by Cytosponge identified 11 of the 13 individuals with active EoE (83%); additional features such as abscesses were also identified. Numbers of eosinophils in samples collected by Cytosponge correlated with those in samples collected by endoscopy (r = 0.50, P = .025). Analysis of tissues collected by Cytosponge identified 4 of the 7 patients without active EoE (57% specificity), as well as 3 cases of active EoE not identified by analysis of endoscopy samples. Including information on level of eosinophil-derived neurotoxin did not increase the accuracy of diagnosis. No complications occurred during the Cytosponge procedure, which was preferred by all patients, compared with endoscopy. CONCLUSIONS: In a feasibility study, the Cytosponge is a safe and well-tolerated method for collecting near mucosal specimens. Analysis of numbers of eosinophils/high-power field identified patients with active EoE with 83% sensitivity. Larger studies are needed to establish the efficacy and safety of this method of esophageal tissue collection. ClinicalTrials.gov number: NCT01585103.

Enhanced innate type 2 immune response in peripheral blood from patients with asthma.

BACKGROUND: In mice, group 2 innate lymphoid cells (ILC2s) likely mediate helminth immunity, inflammation, and tissue repair and remodeling. However, the involvement of ILC2s in human diseases, such as asthma, is not well understood. OBJECTIVES: The goals of this study were to investigate whether peripheral blood specimens can be used to monitor innate type 2 immunity in human subjects and to examine whether ILC2s are involved in human asthma. METHODS: PBMCs from subjects with allergic asthma (AA), subjects with allergic rhinitis (AR), or healthy control (HC) subjects were cultured in vitro with IL-25 or IL-33. Flow cytometry and cell sorting were used to identify, isolate, and quantitate ILC2s in PBMCs. RESULTS: Human PBMCs produced IL-5 and IL-13 when stimulated with IL-33 or IL-25 in the presence of IL-2 without antigens. In addition, IL-7 or thymic stromal lymphopoietin were able to replace IL-2. The cell population with phenotypic ILC2 characteristics, lineage(-)CD127(+)CRTH2(+) cells, responded to IL-33 and produced large quantities of IL-5 and IL-13 but undetectable levels of IL-4. PBMCs from subjects with AA produced significantly larger amounts of IL-5 and IL-13 in response to IL-25 or IL-33 than from subjects with AR or HC. The prevalence of ILC2s in blood was greater in the AA group than in the AR group or the HC group. CONCLUSIONS: Innate type 2 immune responses are increased in asthma but not in AR, suggesting potential differences in the immunopathogenesis of these diseases. Peripheral blood is useful for evaluating innate type 2 immunity in humans.

IL-33 and thymic stromal lymphopoietin mediate immune pathology in response to chronic airborne allergen exposure.

Humans are frequently exposed to various airborne allergens in the atmospheric environment. These allergens may trigger a complex network of immune responses in the airways, resulting in asthma and other chronic airway diseases. In this study, we investigated the immunological mechanisms involved in the pathological changes induced by chronic exposure to multiple airborne allergens. Naive mice were exposed intranasally to a combination of common airborne allergens, including the house dust mite, Alternaria, and Aspergillus, for up to 8 wk. These allergens acted synergistically and induced robust eosinophilic airway inflammation, specific IgE Ab production, type 2 cytokine response, and airway hyperresponsiveness in 4 wk, followed by airway remodeling in 8 wk. Increased lung infiltration of T cells, B cells, and type 2 innate lymphoid cells was observed. CD4(+) T cells and type 2 innate lymphoid cells contributed to the sources of IL-5 and IL-13, suggesting involvement of both innate and adaptive immunity in this model. The lung levels of IL-33 increased quickly within several hours after allergen exposure and continued to rise throughout the chronic phase of inflammation. Mice deficient in IL-33R (Il1rl1(-/-)) and thymic stromal lymphopoietin receptor (Tslpr(-/-)) showed significant reduction in airway inflammation, IgE Ab levels, and airway hyperresponsiveness. In contrast, mice deficient in IL-25R or IL-1R showed minimal differences as compared with wild-type animals. Thus, chronic exposure to natural airborne allergens triggers a network of innate and adaptive type 2 immune responses and airway pathology, and IL-33 and thymic stromal lymphopoietin most likely play key roles in this process.

Airway uric acid is a sensor of inhaled protease allergens and initiates type 2 immune responses in respiratory mucosa.

Although type 2 immune responses to environmental Ags are thought to play pivotal roles in asthma and allergic airway diseases, the immunological mechanisms that initiate the responses are largely unknown. Many allergens have biologic activities, including enzymatic activities and abilities to engage innate pattern-recognition receptors such as TLR4. In this article, we report that IL-33 and thymic stromal lymphopoietin were produced quickly in the lungs of naive mice exposed to cysteine proteases, such as bromelain and papain, as a model for allergens. IL-33 and thymic stromal lymphopoietin sensitized naive animals to an innocuous airway Ag OVA, which resulted in production of type 2 cytokines and IgE Ab, and eosinophilic airway inflammation when mice were challenged with the same Ag. Importantly, upon exposure to proteases, uric acid (UA) was rapidly released into the airway lumen, and removal of this endogenous UA by uricase prevented type 2 immune responses. UA promoted secretion of IL-33 by airway epithelial cells in vitro, and administration of UA into the airways of naive animals induced extracellular release of IL-33, followed by both innate and adaptive type 2 immune responses in vivo. Finally, a potent UA synthesis inhibitor, febuxostat, mitigated asthma phenotypes that were caused by repeated exposure to natural airborne allergens. These findings provide mechanistic insights into the development of type 2 immunity to airborne allergens and recognize airway UA as a key player that regulates the process in respiratory mucosa.

Improving the detection of fungi in eosinophilic mucin: seeing what we could not see before.

OBJECTIVE: To investigate the improvement in histologic detection of fungi with Gomori methenamine silver (GMS) stain by trypsin predigestion in the mucus of patients with chronic rhinosinusitis (CRS). STUDY DESIGN: Prospective, single group, descriptive analysis. SETTING: Multi-institutional. SUBJECTS AND METHODS: Thirty-four sinus specimens from 12 surgical patients with CRS were stained with hematoxylin and eosin, GMS stain, GMS with trypsin digestion, immunofluorescence stains for chitinase, and anti-Alternaria. All patients received skin testing, total IgE serology, and radioallergosorbent tests (RAST) for 23 fungal-specific IgE antibodies. RESULTS: The conventional GMS stain detected fungi in only 9 of 34 (27%) specimens. Predigesting the specimen with trypsin dramatically improved the visualization of fungi (31/34, 91%). The chitinase immunofluorescence visualized fungi in 32 of 34 (94%), and anti-Alternaria visualized 33 of 34 specimens (97%). Only 8 of 12 (75%) patients had detectable allergies. CONCLUSIONS: This report describes a simple modification of the conventional GMS stain that can significantly improve the visualization of fungi on histology and explains the lack of detection in previous studies. These novel, more sensitive histologic methods reveal the presence of fungi within the eosinophilic mucin in allergic and also nonallergic CRS patients, further questioning a crucial role of an IgE-mediated pathophysiology.

Elevated numbers of cells producing interleukin-5 and interleukin-10 in a boy with Kimura disease.

Kimura disease is a rare disorder of unknown etiology, characterized by the presence of benign subcutaneous granuloma, marked peripheral blood eosinophilia and elevation of the immunglobulin E (IgE) serum level. Here, we present a case of a 12-year-old boy with Kimura disease who had a history of repeated severe influenza virus A infection. Along with the characteristic histological findings of granuloma, including eosinophil infiltration, enzyme-linked immunospot assay showed elevated numbers of IL-5- and IL-10-producing cells in the peripheral blood. Immunohistochemical evaluation, however, did not detect IL-5 in the tissue. Possible cytokine dysregulation in Kimura disease was suggested, but the pathogenesis remains unclear.

Increased numbers of eosinophils, rather than only etiology, predict histologic changes in patients with esophageal eosinophilia.

BACKGROUND & AIMS: It can be a challenge to differentiate individuals with eosinophilic esophagitis (EoE) from those with gastroesophageal reflux disease (GERD). We investigated differences in histologic and eosinophil patterns and numbers of mast cells between patients with these disorders. METHODS: We performed histologic analyses and immunohistochemical assays for eosinophil-derived neurotoxin (EDN), major basic protein (MBP), and tryptase, using biopsy samples from 10 patients with GERD (positive results from a pH study and response to proton pump inhibitors), Barrett's esophagus, or EoE (negative results from a pH study and positive response to budesonide). Patients were matched for degree of eosinophilia. RESULTS: Samples from patients with EoE, GERD, or Barrett's esophagus had similar increases in concentrations of eosinophils. Patients with GERD or EoE did not differ in amount of basal zone hyperplasia, microabscesses, spongiosis, eosinophil distribution, maximum eosinophils/high-power field (HPF), or composite histologic scores. Samples from all 3 groups had high levels of EDN and MBP; the levels of eosinophil products were correlated (ρ = 0.93). Extracellular staining for EDN was greater than intracellular staining (2.67 of 3 vs 1.86 of 3); levels tended to be greater in samples from patients with EoE than GERD (P = .05) or Barrett's esophagus (P = .06). Detection of EDN correlated with peak numbers of eosinophils/HPF (ρ = 0.6 for intracellular and extracellular staining). Peak numbers of tryptase-positive mast cells/HPF were significantly greater in samples from patients with EoE than GERD or Barrett's esophagus (P = .01 and .005, respectively). The Spearman correlation between eosinophil and mast cell density was a ρ value of 0.2. CONCLUSIONS: Biopsy samples from patients with GERD and EoE, matched for esophageal eosinophilia, have similar changes in histology and levels of EDN and MBP, whereas mast cells from patients with EoE have higher levels of these products. The presence of esophageal eosinophils, rather than etiology, could be the most important determinant of epithelial response.

IL-33-responsive lineage- CD25+ CD44(hi) lymphoid cells mediate innate type 2 immunity and allergic inflammation in the lungs.

Innate immunity provides the first line of response to invading pathogens and a variety of environmental insults. Recent studies identified novel subsets of innate lymphoid cells that are capable of mediating immune responses in mucosal organs. In this paper, we describe a subset of lymphoid cells that is involved in innate type 2 immunity in the lungs. Airway exposure of naive BALB/c or C57BL/6J mice to IL-33 results in a rapid (<12 h) production of IL-5 and IL-13 and marked airway eosinophilia independently of adaptive immunity. In the lungs of nonsensitized naive mice, IL-33-responsive cells were identified that have a lymphoid morphology, lack lineage markers, highly express CD25, CD44, Thy1.2, ICOS, Sca-1, and IL-7Rα (i.e., Lin(-)CD25(+)CD44(hi) lymphoid cells), and require IL-7Rα for their development. Airway exposure of naive mice to a clinically relevant ubiquitous fungal allergen, Alternaria alternata, increases bronchoalveolar lavage levels of IL-33, followed by IL-5 and IL-13 production and airway eosinophilia without T or B cells. This innate type 2 response to the allergen is nearly abolished in mice deficient in IL-33R (i.e., ST2), and the Lin(-)CD25(+)CD44(hi) lymphoid cells in the lungs are required and sufficient to mediate the response. Thus, a subset of innate immune cells that responds to IL-33 and vigorously produces Th2-type cytokines is present in mouse lungs. These cells may provide a novel mechanism for type 2 immunity in the airways and induction of allergic airway diseases such as asthma.

An 8-year-old boy with hypereosinophilic syndrome.

Hypereosinophilic syndrome (HES) is a heterogeneous group of uncommon disorders characterized by the presence of marked peripheral blood eosinophilia and tissue eosinophilia, resulting in a wide variety of clinical manifestations. We present the case of an 8-year-old boy with HES. He complained of recurrent abdominal pain, general fatigue, and diarrhea. Laboratory data showed marked eosinophilia, elevated total IgE with positive specific IgE antibodies to common inhalant and food allergens, and elevated serum CCL17/TARC. A chest CT scan revealed central bronchiectasis, bronchial wall thickening, a mosaic attenuation pattern, and multiple small nodules in lung parenchyma; abdominal CT showed a thickened bladder wall. Gastrointestinal endoscopy revealed scarring in the gastric mucosa and mucosal erosion in the duodenum. Immunohistochemical examination demonstrated numerous eosinophil infiltrations with extensive extracellular eosinophil major basic protein deposition in the gastric mucosa. Only high-dose oral steroid was effective and cyclosporine appeared to have a steroid-sparing effect. HES is extraordinary rare in children and the long-term prognosis in pediatric HES is not well known. Comprehensive diagnostic procedures are vital for the early detection and management of complications in pediatric HES.

Marked deposition of eosinophil-derived neurotoxin in adult patients with eosinophilic esophagitis.

OBJECTIVES: Eosinophilic esophagitis (EoE) is characterized by infiltration of eosinophils into esophageal epithelium. Blood levels of an eosinophil granule protein, eosinophil-derived neurotoxin (EDN), have been proposed as a biomarker for EoE. However, information regarding localization of EDN in the diseased tissues has not been available. The goal of this study was to evaluate the magnitude and distribution of EDN deposition in tissue specimens from the esophagus of EoE patients. METHODS: We studied specimens from 10 adult EoE patients and eight histologically normal controls (three under age 17). Sections from mid-esophageal biopsy specimens were stained for EDN by immunofluorescence, using a polyclonal rabbit antibody to EDN. Cellular staining (i.e., infiltration of intact eosinophils) and extracellular staining (i.e., deposition of released EDN) were scored in a blinded manner on an established 7-point scale. RESULTS: Esophageal biopsy specimens from histologically normal controls showed no or few intact eosinophils and no or minimal extracellular EDN deposition. In contrast, EDN staining was clearly observed in specimens from all EoE patients. In some EoE patients, marked extracellular EDN deposition was observed despite relatively small numbers of intact eosinophils. Overall, there was no correlation between the eosinophil infiltration and the extracellular EDN staining scores. CONCLUSIONS: Marked tissue deposition of extracellular EDN is present in the esophagus of EoE patients. Tissue eosinophil counts may underestimate how extensively eosinophils are involved, particularly in individuals with marked eosinophil degranulation. Evaluation of EDN staining in esophageal biopsy specimens may be useful to diagnose and manage patients with EoE.

Beta2-integrin-mediated adhesion and intracellular Ca2+ release in human eosinophils.

Human eosinophils spontaneously adhere to various substrates in the absence of exogenously added activators. In the present study a method was developed for characterizing eosinophil adhesion by measuring changes in impedance. Impedance measurements were performed in HCO(3)-buffered HybriCare medium maintained in a humidified 5% CO(2) incubator at 37 degrees C. Impedance increased by more than 1 kOmega within minutes after eosinophils made contact with the substrate, reaching a peak within 20 min. Blocking mobilization of intracellular [Ca(2+)] that precedes adhesion with BAPTA-AM (10 microM) completely inhibited the rise in impedance as well as the changes in cell shape typically observed in adherent cells. However, lowering the extracellular [Ca(2+)] with 2.5 mM EGTA did not inhibit the increase in impedance. Pretreatment with anti-CD18 antibody to block substrate interactions with beta(2)-integrins, or jasplakinolide (2 microM) to block actin reorganization, abolished the increase in impedance and adherent morphology of the cells. Exposure of eosinophils to the phosphatidylinositol 3 kinase inhibitor LY294002 (5 microM) or treatment with protein kinase C zeta pseudosubstrate to competitively inhibit activity of the enzyme significantly reduced the increase in impedance and inhibited the cell spreading associated with adhesion. These results demonstrate a novel method for measuring eosinophil adhesion and showed that, following formation of a tethered attachment, a rapid increase in intracellular [Ca(2+)] precedes the cytoskeletal rearrangements required for cell shape changes and plasma membrane-substrate interactions associated with adhesion.

Major basic protein homolog (MBP2): a specific human eosinophil marker.

Human eosinophil granule major basic protein (MBP1) is an exceedingly basic (isoelectric point >11) 14-kDa protein, comprising the core of the secondary eosinophil granule. Recently, a less cationic homolog of MBP, termed MBPH or simply, MBP2, has been discovered. We prepared a panel of mAbs to MBP2 and used these Abs to localize and quantitate this molecule in leukocytes and biological fluids. Specific mAbs for MBP2 were selected using slot-blot analyses and used in a two-site immunoassay, Western blotting, and immunofluorescence microscopy. The sensitivity of the immunoassay was markedly improved by reduction and alkylation of MBP2. MBP1 is more abundant than MBP2 in lysates of eosinophils and their granules, as judged by immunoassay and Western blotting. By immunofluorescence, MBP1 is present in eosinophils, basophils, and a human mast cell line (HMC1), whereas MBP2 is only detected in eosinophils. Neither MBP1 nor MBP2 could be detected in any other peripheral blood leukocyte. MBP2 levels measured in plasma and serum were essentially identical. In contrast to past measurements for MBP1, MBP2 was not detected above normal levels in sera from pregnant donors. However, measurement of serum MBP2 discriminated patients with elevated eosinophils from normal subjects, and MBP2 was also detectable in other biological specimens, such as bronchoalveolar lavage, sputum, and stool. These results indicate that MBP2 is present only in eosinophils and that it may be a useful biomarker for eosinophil-associated diseases.

Eosinophilic oophoritis: association with positive Strongyloides stercoralis serology and clinical response to ivermectin.

BACKGROUND: Localized eosinophilic infiltration causing ovarian dysfunction is an unusual clinical problem. CASE: We report a 16-year-old girl with abdominal pain, ovarian cysts, and biopsy-proven eosinophilic oophoritis, which resulted in right oophorectomy. Subsequently, a large cyst developed in the left ovary, causing abdominal pain that was worse at the time of her menses, and biopsy again showed numerous eosinophilic microabscesses. Unexpectedly, a parasite serology test to Strongyloides stercoralis was positive, although stool tests for ova and parasites were negative and the total IgE and total eosinophil count were normal. After treatment with ivermectin, the patient's abdominal pain resolved, the serologic antibody titers to S stercoralis returned to normal, and subsequent ultrasonographic evaluations showed involution of the large cyst in the remaining ovary. CONCLUSION: Eosinophilic oophoritis is a new disorder of localized tissue eosinophilia.

The role of ubiquitous airborne fungi in chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is a confusing disease for both allergists and otorhinolaryngologists, partly because of its poorly understood pathophysiology and partly because of its limited treatment options. Several recent reports have provided evidence for a better understanding of the etiology and the relationship of CRS to airborne fungi-especially to Alternaria. First, the development of novel methods enables detection of certain fungi in mucus from the nasal and paranasal sinus cavities. Second, a non-IgE-mediated immunological mechanism for reactivity of patients with CRS to certain common fungi has been described. Third, these fungi are surrounded by eosinophils in vivo, suggesting that they are targeted by eosinophils. Finally, the preliminary results of studies using antifungal agents to treat patients with CRS are promising. Overall, these recent discoveries provide a logical mechanism for the pathophysiology of CRS, and they also suggest promising avenues for treatment of CRS with antifungal agents.

Identification of basophils by a mAb directed against pro-major basic protein 1.

BACKGROUND: Basophils possess characteristics of both mast cells and eosinophils, and all 3 cell types often are found together, particularly during allergic reactions. A mAb (J175-7D4) generated against the recombinant pro-form of human eosinophil granule major basic protein 1 (rproMBP1) appeared to stain only basophils in tissue specimens. OBJECTIVE: We investigated J175-7D4 to characterize its specificity for basophils. METHODS: Fluid-phase immunoprecipitation, Western blotting, and immunocytochemistry and immunohistochemistry were used to establish the specificity of J175-7D4. RESULTS: First, J175-7D4 binds to various glycosylated and proteolytically processed forms of rproMBP1, but not to major basic protein. Second, cells transfected with the rproMBP1 gene and human placental tissue (known to express the pro-form of major basic protein 1 [proMBP1]) stain specifically with J175-7D4. In contrast, although mature eosinophils contain substantial major basic protein, they lack proMBP1 and do not stain. Neutrophils, lymphocytes, monocytes, and skin mast cells also are not stained. However, blood basophils are stained by J175-7D4, anti-IgE, Wright-Giemsa (metachromatically), and a previously characterized basophil-specific mAb, 2D7. Finally, formaldehyde-fixed, paraffin-embedded basophils are identically detected by J175-7D4 and 2D7, and J175-7D4 also recognizes putative basophils in formaldehyde-fixed, paraffin-embedded tissue specimens from inflammatory dermatoses, such as atopic dermatitis and delayed pressure urticaria. CONCLUSION: The J175-7D4 mAb recognizes proMBP1 as a novel marker for human basophils. J175-7D4 should prove useful for characterizing basophil involvement in human health and disease.

The role of ubiquitous airborne fungi in chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is a confusing disease for both allergists and otorhinolaryngologists, partially due to its poorly understood pathophysiology and partially due to its limited treatment options. Several recent reports now provide evidence for a better understanding of the etiology and the relationship of CRS to airborne fungi, especially to Alternaria. First, the development of novel methods enables detection of certain fungi in mucus from the nasal and paranasal sinus cavities. Second, a non-immunoglobulin E-mediated immunologic mechanism for reactivity of CRS patients to certain common fungi has been described. Third, these fungi are surrounded by eosinophils in vivo, suggesting that they are targeted by eosinophils. Fourth, the preliminary results of studies using antifungal agents to treat patients with CRS are promising. Overall, these recent discoveries provide a logical mechanism for the pathophysiology of CRS, and they also suggest promising avenues for treatment of CRS with antifungal agents.

Striking deposition of toxic eosinophil major basic protein in mucus: implications for chronic rhinosinusitis.

BACKGROUND: The mechanisms by which eosinophilic inflammation damages the epithelium and contributes to recurrent acute exacerbations in chronic rhinosinusitis (CRS) have not been fully elucidated. OBJECTIVE: We tested the hypotheses that eosinophils deposit toxic major basic protein (MBP) in the mucus and that MBP reaches concentrations able to damage the sinonasal epithelium. METHODS: Tissue specimens with mucus attached to the tissue were carefully collected from 22 patients with CRS and examined by using immunofluorescence staining for MBP. This immunofluorescence was digitally analyzed to determine the area covered by MBP and the intensity of the staining (estimating MBP concentration). Levels of MBP in extracts from nasal mucus were quantitated by means of RIA. RESULTS: Heterogeneous eosinophilia was evident within tissue and mucus specimens. All tissue specimens showed intact eosinophils, but diffuse extracellular MBP deposition, as a marker of eosinophil degranulation, was rare. In contrast, all mucus specimens showed diffuse MBP throughout and abundant diffuse extracellular MBP deposition within clusters of eosinophils. Digitized analyses of MBP immunofluorescence revealed increased area coverage (P < .0001) in mucus compared with that seen in tissue. Estimated concentrations of MBP within the clusters suggested toxic levels. MBP concentrations in mucus extract reached 11.7 microg/mL; MBP was not detectable in healthy control subjects. CONCLUSION: In patients with CRS, eosinophils form clusters in the mucus where they release MBP, which is diffusely deposited on the epithelium, a process not observed in the tissue. Estimated MBP levels far exceed those needed to damage epithelium from the luminal side and could predispose patients with CRS to secondary bacterial infections.